In order to understand the role of protein-bound carbohydrates in nature and in pathological processes, we have further documented the observation that these structures can change in a programmed manner. We have found that mouse teratocarcinoma cells (F9) produce fibronectin of Mr\= 250,000 containing covalently bound heparin sulfate and lactosaminoglycan. Upon differentiation to become the FAACC19 cell, it continues to make fibronectin, but the Mr = 220,000, and it lacks the two carbohydrate polymers. Attempts are being made to determine how the cell can effect this genetically programmed change by examining the levels of various enzymes involved in the metabolism of carbohydrates. We have found that the sialic acid (NAN) content of the differentiated cell is double that of the undifferentiated cell and that sialyl transferase is increased 5-\to 9-fold. This increased sialylation of growing carbohydrate chains could prevent the synthesis of the lactosylaminoglycans. We have also found that the enzyme that cleaves sialic acid into pyruvate and N-acetylmannosamine and could control the level of sialic acid in the cell rises 10-fold upon differentiation of F9 into PyS cells and at least 80-fold within 2 days in the promyelocytic leukemic cell, HL60, that is induced to differentiate into a macrophage by phorbol ester. The level goes up several-fold in growing cells and drops precipitously when cells stop dividing (manuscript in preparation). The level decreases 2-\to 4-fold in four sets of malignant cells tested. At least 30% of the activity is in the nucleus, the locus of the sialic acid-activating enzyme. On the other hand, the rates of biosynthesis of NAN and its activation (to CMPNAN) are approximately the same in F9 and PyS. This suggests that control of the level of sialic acid in a cell may reside in the rate of its degradation. We have observed significant changes in the rates of degradation of activated sugars (UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose) with growth, differentiation, and malignant transformation. These changes are being studied systematically. Studies are being carried out demonstrating the influence of environment, drugs, and composition of culture medium on the carbohydrate structures of the IgG produced by hybridoma cells. We have found that the carbohydrates of fibronectin of "old" cells differ from those of "young" cells. These studies are aimed at demonstrating and delineating the mechanisms of changes induced in protein-bound carbohydrates by genetic programming and by environmental influences. (A)